Maternal protein restriction may be a risk factor for cardiovascular disorders in adulthood. The RAS (renin-angiotensin-system) plays a pivotal role in cardiac remodeling. Components of the RAS, including angiotensin II (AngII) and its receptors type 1 (AT1R) and 2 (AT2R) are expressed in the heart. This study investigates whether gestational protein restriction alters the expression and localization of AT1R and AT2R and RAS signaling pathway proteins in parallel with left ventricle hypertrophy and systemic hypertension in male offspring. Dams were kept on normal (NP, 17% protein) or low (LP, 6% protein) protein diet during pregnancy. Systolic blood pressure (SBP) of male offspring was measured from the 8 th to 16 th week and left ventricles of 16-wk-old rats were processed for histology, morphometric, immunoblotting and immunohistochemistry. LP offspring showed a significant reduction in birth body weight and SBP increased significantly from the 8 th week. Left ventricle mass and cardiomyocytes area were also significantly higher in LP animals. Widespread perivascular fibrosis was not detected in the heart tissue. Analysis by immunoblotting and immunohistochemistry demonstrated a significant enhance in cardiomyocyte expression of AT1R and ERK1 in LP offspring. Expression of PI3K in LP was significantly reduced in cardiomyocytes and in the intramural coronary wall, while AT2R expression was unchanged in the NP group. We also found reduced LP expression of JAK2 and STAT3. In conclusion, our data also suggest that changes in the RAS may play a role in the ventricular growth through upregulation of the AT1-mediated ERK1/2 response, despite unchanged AT2R expression.
Nutritional alterations and fetal endogen patterns lead to the development of physiological and metabolic changes, predisposing the individual to metabolic, endocrine and cardiovascular diseases in adult life [
The experiments were conducted on age-matched, female offspring of sibling-mated Wistar Hannover rats. The general guidelines established by the Brazilian College of Animal Experimentation (COBEA) were followed throughout the investigation. Our local colonies originated from a breeding stock supplied by CEMIB/UNICAMP, Campinas, SP, Brazil. Ten females were mated and were maintained on isocaloric standard rodent laboratory with normal protein content [NP, n = 5] (17% protein) or low protein content [LP, n = 5] (6% protein) chow ad libitum intake throughout the entire pregnancy. All groups returned to isocaloric standard chow intake after delivery. For the experiments, we used male pupsfrom randomized dams [8,9].
The systolic blood pressure (SBP) was measured in conscious male offspring at 8 to 16 weeks of age, employing an indirect tail-cuff method using an electrosphygmomanometer combined with a pneumatic pulse transducer/amplifier (IITC Life Science). This indirect approach allowed repeated measurements with a close correlation (correlation coefficient = 0.975), compared to direct intra-arterial recording [10,11]. The mean of three consecutive readings represented the blood pressure. All the maleoffspring were used.
Sixteen-week-old male offspring from the NP (n = 5) and LP (n = 5) groups were anesthetized with ketamine (75 mg∙kg−1 body weight, i.p.) and xylasine (10 mg∙kg−1 body weight, i.p.). The animals were perfused by the left carotid artery with saline containing heparin (5%) for 15 min. Then followed by perfusion with 0.1 M phosphate buffer (pH 7.4) containing 4% (w/v) paraformaldehyde for 25 min. After perfusion, the cardiac left ventricles were removed and representative samples were fixed in 4% phosphate-buffered formalin during 24 h for paraffin embedding. Five-micrometer-thick sections were cut from the blocked tissue and stained with hematoxylin-eosin. Cardiomyocytes cross-sectional area was determined for at least 100 myocytes per slide stained with hematoxylin-eosin. The measurements were performed using a Leica microscope (×40 magnification lens) attached to a video camera and connected to a personal computer equipped with image analyzer software (Image Pro Express 6.0, Media Cybernetics, Inc.). Cardiomyocyte area was measured with a digitizing pad, and the selected cells were transversely cut with the nucleus clearly identified in the center of the myocyte. Other sections were stained with Sirius Red to evaluate interstitial and perivascular fibrosis. For immunohistochemical analysis we use anti-AT1R, AT2R, ERK1/2, PI3K, JAK2 and STAT3 antibodies (Santa Cruz Biotech, Inc., CA, USA). Antigen retrieval was performed using 0.01 M citrate buffer (pH 6.0) boiling in microwave oven (1.300 W) twice for 5 min each. Proteins were immunohistochemically detected using the avidin-biotin-peroxidase method. Briefly, deparaffinized 5-μm-thick heart sections on poly-l-lysine coated slides were treated with 3% H2O2 in phosphatebuffered saline for 15 min, nonfat milk for 60 min, primary antibodies for 60 min, and avidin-biotin-peroxidase solution (Vector Laboratories Inc., CA, USA, 1:1:50 dilution). Chromogen color was accomplished with 3,3’- diaminobenzidine tetrahydrochroride (DAB, Sigma-Aldrich Co., St. Louis MO, USA) as the substrate to demonstrate the sites of peroxidase binding. The slides were counterstained with Harris’s hematoxylin.
Sixteen-week-old male offspring rat from the NP (n = 5) and LP (n = 5) groups had their neck dislocated, and the abdominal cavity was opened for cardiac left ventricle removal. The tissue was minced coarsely and homogenized immediately in 10 volumes of solubilization buffer (10 ml/L Triton-X 100, 100 mM Tris [hydroxymethyl]amino-methane (Tris) pH 7.4, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM ethylendiaminetetracetic acid (EDTA), 10 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride (PSMF) and 0.1 mg/ml aprotinin at 4˚C, using a polytron PTA 20S generator (model PT 10/35, Brinkmann Instruments, Westbury, NY, USA) operated at maximum speed for 20 s. The tissue extracts were centrifuged at 11.000 rpm at 4˚C for 40 min, and the supernatants used as sample.
Protein quantification was performed using the Bradford method. For quantification, both tissue and total extract samples (250 mg protein) were subjected to SDSPAGE. After electrophoretic separation, proteins were transferred to nitrocellulose membranes and then blotted with specific antibody. The samples were treated with Laemmli buffer containing 100 mM dithiothreitol (DTT), heated in a boiling water bath for 4 min and subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a Bio-Rad minigel apparatus (Mini-Protean, Bio-Rad). Electrotransfer of proteins from the gel to the nitrocellulose membranes was performed for 90 min at 120 V (constant) in a Bio-Rad miniature transfer apparatus (Mini-Protean). The non-specific protein binding to the nitrocellulose was reduced by preincubating the filter for 2 h at 22˚C in blocking buffer (5% non-fat dry milk, 10 mM Tris, 150 mM NaCl, and 0.02% Tween 20). The nitrocellulose blots were incubated at 4˚C overnight with primary antibodies diluted in blocking buffer (3% non-fat dry milk, 10 mM Tris, 150 mM NaCl, and 0.02% Tween 20). Immunoreactive bands were detected usingthe enhanced chemiluminescence method (RPN 2108 ECL Westernblotting analysis system; Amersham Biosciences) and were detected by autoradiography using preflashed Kodak XAR film (Eastman Kodak, Rochester, NY) with Cronex Lightning Plus intensifying screen (DuPont, Wilmington, DE) for 10 min. Images of the developed radiographs were scanned (Epson Stylus 3500) and band intensities were quantified by optical densitometry (Scion Image Corporation). To ensure equal loading, membranes were stained with reversible Ponceau to discard possible inequalities in protein loading and/or transfer, in western blots [
All numerical results are expressed as the mean ± SEM of the indicated number of experiments. Data obtained over time were analyzed using one-way ANOVA. Post-hoc comparisons between selected means were performed with Bonferroni’s contrast test when initial ANOVA indicated statistical differences between experimental groups. Comparisons involving only two means within or between groups were carried out using a Student’s t test. The results of blots are presented as direct comparisons of bands in autoradiographs and quantified by densitometry using the Scion Image software (ScionCorp). The level of significance was set at P ≤ 0.05.
The birth weight of the LP male pups was significantly reduced when compared to NP male pups (6.15 ± 0.16 g vs. 6.72 ± 0.41 g respectively, P = 0.008). Systolic blood pressure (SBP) was significantly higher in LP than in NP rats from 8 to 16 weeks of age (
Western blot analysis in male offspring of NP and LP cardiac left ventricle yielded a single band at the expected weight of corresponding proteins. Heart AT2R expression was unchanged when compared to NP group (NP: 12.14 ± 0.09 vs. LP: 12.02 ± 0.72, P = 0.8, Figure
4). Analysis by immunoblotting, confirmed by immunohistochemistry, demonstrated a significantly augmented
cardiomyocyte expression of AT1R in LP offspring (NP: 12.27 ± 1.38 vs. LP: 19.98 ± 1.66, P = 0.01, Figures 4 and 5) and ERK1 (NP: 14.03 ± 0.064 vs. LP: 16.23 ± 0.063, P = 0.001, Figures 4 and 5). On the other hand, the expression of PI3K in LP was significantly reduced in cardiomyocytes and in the intramural coronary wall (NP: 20.52 ± 0.79 vs. LP: 11.21 ± 0.34, P = 0.008, Figures 4 and 5). By immunohistochemical analysis we verified that LP expression of JAK2 and STAT3 are reduced in both cardiomyocytes and the coronary endothelium (
The hypothesis that a fetus may control its own development according to the intrauterine environment suggests that the offspring adjusts its growth and metabolism. The imbalance between food intakes, homeostasis and energy consumption leads to an increased risk of cardiovascular and metabolic diseases [8,9,13,14]. Herein, we report the development of adult hypertension, in as-
sociation with left ventricle morphological changes, in a maternal undernutrition offspring model.
The effect of the reduced birth weight was associated with a significant and progressive augmentation in arterial blood pressure in the LP group in parallel with increased left ventricular mass and volume. However, the main mechanisms link an intrauterine adverse environment with the development of hypertensionremain unknown. Animal models of fetal programming induced by gestational protein undernutrition or placental insufficiency report common temporal alterations in the RAS [8,9,15]. Most of the known physiological effects of AngII are mediated by AT1R, which serve as a control point for regulating the ultimate effects of AngII on its target tissue. In the present study, expression of AT1R was upregulated despite unchanged AT2R expression in the left ventricular maternal 16-wk-old LP offspring, suggesting that the RAS could play a role in fetal heart enlargement following maternal underfeeding. In adult hearts, AngII appears to cause fibrosis and hypertrophy [
The present data confirms previous studies [
In the current study, the increased expression of AT1R, associated with the activation of the JAK/STAT mitogenic pathway, was not confirmed as we showed a reduction of JAK2 and STAT3 expression. Accumulating evidence supports PI3-kinase’s role in the tyrosine phosphorylation of STATs [
In conclusion, the current study confirms experimental and epidemiological studies indicating that maternal underfeeding is associated with low birth weight offspring and may result in an increased risk of cardiovascular morbidity in adulthood. Our data also suggest that changes in the RAS may play a role in the ventricular growth through upregulation of the AT1-mediated ERK1/2 response, despite unchanged AT2R expression. In addition, the findings of present study show that maternal underfeeding did not change interstitial structures in adult hearts. Although compelling evidence has been reported to implicate RAS signaling pathways in a variety of myocardial responses, additional studies are required to firmly test these statements and establish its importance in programming disease processes in the heart.
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (Proc. 05/54362-4 and 10/52696-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.